Regulation of miRNA function happens at the first step of its biogenesis. Reduced enzymatic activity of Drosha has been described in various malignancies. Applying CRISPR-Cas9 based genome-wide loss-of-function screen, we seek to identify novel cellular regulators of Drosha activity. To this end, we inserted pri-miR-16 sequence into the 3'UTR of the mCherry reporter, making the reporter mRNA a target of Drosha cleavage. Stable cell lines were established in which such reporter was co-expressed with an eYFP control. After normalizing to eYFP, the signal of mCherry serves as a faithful indicator of Drosha activity in cells. As expected, reporters containing pri-miR-16 exhibited robust de-repression of mCherry upon Drosha Knockout. We randomly knocked out cellular genes via a treatment of CRISPR-Cas9 library. Cells in which Drosha activity was impaired were sorted out based on the ratio of mCherry to eYFP. sgRNA representation in the sorted and unsorted cells was enumerated by targeted high-throughput sequencing of the genomic DNA. HiTSelect was used to analyze combined results of 4 independent screens, generating high-confidence hits. The well-known components of pri-miRNA processing - Drosha and DGCR8, ranked top of the results, establishing specificity of the screen. Several novel factors were selected for further validation. Loss-of-function of these genes de-repressed the pri-miR-16 mCherry reporter, and dramatically reduced mature miR-16 expression. Interestingly, such effect is limited to a subset of endogenous miRNA, indicating miRNA-specific regulation. Together, our results reveal a large number of previously unknown regulators of microprocessor and provide insights into understanding miRNA biogenesis regulations in vivo. Based mainly on non-cellular experiments, the current model posits that Drosha's activity is restrained inside the nucleus. By studying Drosha cleavage in living cells, we gather evidence to establish the existence of cytoplasmic Drosha (c-Drosha) activity. More importantly, we reveal alternative splicing as an underlying mechanism to induce c-Drosha activity. We identify a novel Drosha isoform in which the exon containing the putative nuclear localization signal (NLS) is skipped. This isoform is abundant in multiple cell lines and its levels vary dramatically among different human tissues, suggesting that c-Drosha has unique biological functions in gene regulation. Further analysis indicates that c-Drosha expression is upregulated in multiple cancer tissues, raising an intriguing possibility that c-Drosha functions as an oncogene by targeting mRNAs of tumor-suppressor genes for degradation. We have collaborated with Dr. Markus Hafner (NIAMS) to identify the direct targets of c-Drosha to further investigate c-Drosha's role in cancer development. Several novel targets were found. We are in middle of validating these exciting findings. MicroRNAs (miRNA) maturation starts with the Drosha cleavage, the fidelity of which is critical for the downstream processing and determines miRNA's target specificity. To understand how the structure of the pri-miRNA impacts on the biogenesis of miRNAs, we studied the maturation of the three mir-9 paralogs. We find that bulges bending the tertiary structure of pri-mir-9-1 foster alternative cleavages. This generates a novel miRNA - miR-9-alternative, which has a shifted seed targeting a unique set of genes compared to canonical miR-9. Further analysis reveals that miR-9-alternative plays a distinct role in low grade gliomas. The study of tertiary structures of pri-miRNAs suggest that coaxial bending of pri-miRNAs could be a general mechanism generating novel miRNA isomer (isomiR) with functional and evolutionary importance. Finally, we have applied the insights gained from the study of miRNA biogenesis into designing new generation of shRNAs. Currently, unsatisfactory knockdown efficacy and off-target effects hamper shRNA applications, often due to inefficient,low fidelity of processing. Our previous finding on Dicer processing has established a loop-counting rule, which laid the groundwork in designing Pol III-driven pre-miRNA-like shRNAs free of the off-target effects resulting from heterogeneous processing. We are currently working on transferring such a design into the Pol II system, in which more complicated manipulation of shRNA function is possible. In particular, we are developing conditionally activated shRNAs whose function can be specifically turned on in cancer cells. This approach will dramatically increase shRNA specificity and safety. Given the long half-lives of mature miRNAs (ranging from hours to days), biogenesis control by itself is inadequate in situations that require rapid changes in miRNA function. Post-maturation regulation is an important component of how miRNAs function. Thus, in alignment with our goal of understanding miRNA regulation, we study the biogenesis and function of 3' isomiRs, which are miRNA variants generated by post-maturation tailing (adding nucleotides) and/or trimming (removing nucleotides). To investigate the function of 3' isomiR, we checked the status of 3' sequence modifications during miRNA overexpression and decay. We found that isomiR profiles are not random but rather tightly regulated. This suggests that the 3' end modification is not merely a consequence of miRNA overexpression, but rather plays an active role in maintaining the miRNA hemostasis. To establish a causative relation, we are working on identifying the enzymes that are responsible for producing certain isomeric forms. We also aim to develop novel strategies to monitor the function specific to certain isomiRs. Overall, these studies seek to significantly advance our basic understanding of isomiRs, and provide a foundation for future mechanistic study of their functions in cancer.